Evaluation and Selection of Bacillus Species Based on Enzyme Production, Antimicrobial Activity, and Biofilm Synthesis as Direct-Fed Microbial Candidates for Poultry

Social concern about misuse of antibiotics as growth promoters (AGP) and generation of multidrug-resistant bacteria have restricted the dietary inclusion of antibiotics in livestock feed in several countries. Direct-fed microbials (DFM) are one of the multiple alternatives commonly evaluated as substitutes of AGP. Sporeformer bacteria from the genus <i>Bacillus</i> have been extensively investigated because of their extraordinary properties to form highly resistant endospores, produce antimicrobial compounds, and synthesize different exogenous enzymes. The purpose of the present study was to evaluate and select <i>Bacillus</i> spp. from environmental and poultry sources as DFM candidates, considering their enzyme production profile, biofilm synthesis capacity, and pathogen-inhibition activity. Thirty-one <i>Bacillus</i> isolates were screened for <i>in vitro</i> relative enzyme activity of amylase, protease, lipase, and phytase using a selective media for each enzyme, with 3/31 strains selected as superior enzyme producers. These three isolates were identified as <i>Bacillus subtilis</i> (1/3), and <i>Bacillus amyloliquefaciens</i> (2/3), based on biochemical tests and 16S rRNA sequence analysis. For evaluation of biofilm synthesis, the generation of an adherent crystal violet-stained ring was determined in polypropylene tubes, resulting in 11/31 strains showing a strong biofilm formation. Moreover, all <i>Bacillus</i> strains were evaluated for growth inhibition activity against <i>Salmonella enterica</i> serovar Enteritidis (26/31), <i>Escherichia coli</i> (28/31), and <i>Clostridioides difficile</i> (29/31). Additionally, in previous <i>in vitro</i> and <i>in vivo</i> studies, these selected <i>Bacillus</i> strains have shown to be resistant to different biochemical conditions of the gastrointestinal tract of poultry. Results of the present study suggest that the selection and consumption of <i>Bacillus</i>-DFM, producing a variable set of enzymes and antimicrobial compounds, may contribute to enhanced performance through improving nutrient digestibility, reducing intestinal viscosity, maintaining a beneficial gut microbiota, and promoting healthy intestinal integrity in poultry.